Effects of Hst3p inhibition in Candida albicans: a genome-wide H3K56 acetylation analysis

Candida spp. represent the third most frequent worldwide cause of infection in Intensive Care Units with a mortality rate of almost 40%. The classes of antifungals currently available include azoles, polyenes, echinocandins, pyrimidine derivatives, and allylamines. However, the therapeutical options for the treatment of candidiasis are drastically reduced by the increasing antifungal resistance. The growing need for a more targeted antifungal therapy is limited by the concern of finding molecules that specifically recognize the microbial cell without damaging the host. Epigenetic writers and erasers have emerged as promising targets in different contexts, including the treatment of fungal infections. In C. albicans, Hst3p, a sirtuin that deacetylates H3K56ac, represents an attractive antifungal target as it is essential for the fungus viability and virulence. Although the relevance of such epigenetic regulator is documented for the development of new antifungal therapies, the molecular mechanism behind Hst3p-mediated epigenetic regulation remains unrevealed. Here, we provide the first genome-wide profiling of H3K56ac in C. albicans resulting in H3K56ac enriched regions associated with Candida sp. pathogenicity. Upon Hst3p inhibition, 447 regions gain H3K56ac. Importantly, these genomic areas contain genes encoding for adhesin proteins, degradative enzymes, and white-opaque switching. Moreover, our RNA-seq analysis revealed 1330 upregulated and 1081 downregulated transcripts upon Hst3p inhibition, and among them, we identified 87 genes whose transcriptional increase well correlates with the enrichment of H3K56 acetylation on their promoters, including some well-known regulators of phenotypic switching and virulence. Based on our evidence, Hst3p is an appealing target for the development of new potential antifungal drugs

Biallelic RIPK1 mutations in humans cause severe immunodeficiency, arthritis, and intestinal inflammation.

RIPK1 (receptor-interacting serine/threonine kinase 1) is a master regulator of signaling pathways leading to inflammation and cell death and is of medical interest as a drug target. We report four patients from three unrelated families with complete RIPK1 deficiency caused by rare homozygous mutations. The patients suffered from recurrent infections, early-onset inflammatory bowel disease, and progressive polyarthritis. They had immunodeficiency with lymphopenia and altered production of various cytokines revealed by whole-blood assays. In vitro, RIPK1-deficient cells showed impaired mitogen-activated protein kinase activation and cytokine secretion and were prone to necroptosis. Hematopoietic stem cell transplantation reversed cytokine production defects and resolved clinical symptoms in one patient. Thus, RIPK1 plays a critical role in the human immune system

Study of bio-molecar interaction: a) Mapping of the interaction between STAT1 and flavonoids; b) Role of PDIA6-BiP complex in the regulation of the unfolded protein response

2010 - 2011Mapping of the interaction between STAT1 and flavonoids Abstract An experimental approach is described, in the first part of this Ph.D. work, for determining protein-small molecule non-covalent ligand binding sites and protein conformational changes induced by ligand binding. The methodology utilizes a combination of multiple technical approaches: limited proteolysis, MALDI TOF MS, circular dichroism and Surface Plasmon Resonance (SPR) to determine the binding sites in signal transducer and activator of transcription 1, STAT1 (87kDa)-flavonoid (Epigallocatechin-3-gallate, Myricetin and Delphinidin, about 500 Da) non-covalent complex. Comparing relative ion abundances of peptides released from the limited proteolysis of STAT1 and the STAT1-flavonoid complex after 0, 5, 15 and 30 minutes of digestion revealed that the binding of flavonoid induced a significant change in surface topology of STAT1. An increase in ion abundance and a different peptide profile suggest that the flavonoids obstruct the access of the proteases to one or both termini of specific peptides, identifying flavonoids binding region. Taken together, MALDI MS and SPR data led us to assume that the binding sites are close to Tyrosine 701 and that the flavonoids probably act disturbing the phosphorylation of TYR701 and the following dimerization and activation of STAT1. PDIA6-BiP complex: role in the regulation of the unfolded protein response Abstract The unfolded proteins response (UPR) induced in many experimental settings is an extremely strong response that usually leads to cell death rather than to restoration of the ER homeostasis. Because the outcome of UPR signaling determines cell fate, a key unresolved molecular question is how UPR signaling is attenuated. Indeed, it is often under-appreciated that UPR signaling in response to stress is transient and is attenuated. Recently it has been proved that yeast UPR matches its output to the magnitude of the stress by regulating the duration of IRE1 signaling. An ER protein, known as binding immunoglobulin protein (BiP), binding to UPR sensors regulates their deactivation. Our idea, described in the second part of this Ph.D. work, is that there is another luminal ER factor, which interacts with the UPR sensors and is involved in attenuation of their activities. This factor is protein disulphide isomerase 6, PDIA6 (also known as P5), a poorly understood member of the protein disulfide isomerase (PDI) family, whose absence, according to our data, confers hypersensitivity to ER stress because one of its main action is tied to the sensing of UPR, rather than to the consequences of UPR signaling. We thought that PDIA6 uses its protein disulfide isomerase activity to interact specifically with UPR sensors in the ER lumen and attenuate their activities, thus regulating the duration of ER stress signaling. [edited by author]X n.s

Editorial on: Genetic Determinants and Prediction of Antibiotic Resistance Phenotypes in Helicobacter pylori

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A new serotonin 2A receptor antagonist with potential benefits in Non-Alcoholic Fatty Liver Disease

Peripheral 5-hydroxytryptamine 2A receptor (5-HT2AR) could be a new pharmacological target for NASH, an evolution of NAFLD characterized by hepatic steatosis, cytoskeletal alterations, and hepatic inflammation that can arise with or without fibrosis. SJT4a is a synthetic β-carboline antagonist for 5-HT2AR developed by SJT molecular research to treat NASH. We performed a combined in silico/in vivo study on this potential drug to elucidate its activity and possible mechanism of action. The in silico protocol compares SJT4a with four known 5-HT2AR ligands with different activities (LSD, methiothepin, zotepine, risperidone). We performed molecular docking calculations, evaluation of binding energy by AI-based methods and Molecular Dynamics simulations of the five ligand-target complexes. Moreover, we used a pseudo-semantic analysis to evaluate the potential mechanism of action of SJT4a. In silico predictions and pseudo-semantic analysis suggested antagonistic activity for SJT4a. The in silico prediction was confirmed by [3H]-5HT radioligand binding together with SJT4a competition analysis in CHO-K1 cell cultures expressing 5-HT2AR. SJT4a was then tested in vivo. We investigated the effect of 8 weeks of treatment with SJT4A on metabolic parameters, liver pathology, NAFLD activity score, and fibrosis stage in male DIO-NASH C57BL/6 J mice diet-induced obesity fed with an obesogenic diet compared with DIO-NASH and LEAN-CHOW vehicles. In our tests, SJT4a showed intense activity in diminishing the most relevant hallmarks of NASH in the DIO-NASH mice model. We proposed a possible mode of action for SJT4a based on its 5-HT2AR antagonist activity

Helicobacter pylori Pathogen-Associated Molecular Patterns: Friends or Foes?

Microbial infections are sensed by the host immune system by recognizing signature molecules called Pathogen-Associated Molecular Patterns—PAMPs. The binding of these biomolecules to innate immune receptors, called Pattern Recognition Receptors (PRRs), alerts the host cell, ac- tivating microbicidal and pro-inflammatory responses. The outcome of the inflammatory cascade depends on the subtle balance between the bacterial burn and the host immune response. The role of PRRs is to promote the clearance of the pathogen and to limit the infection by bumping inflammatory response. However, many bacteria, including Helicobacter pylori, evolved to escape PRRs’ recognition through different camouflages in their molecular pattern. This review examines all the different types of H. pylori PAMPs, their roles during the infection, and the mechanisms they evolved to escape the host recognition

Gastric TFF1 Expression from Acute to Chronic Helicobacter Infection

TFF1, a mucin-associated secreted peptide of gastric mucous cells, is known as a protective agent for stomach epithelium under different stimuli, but its role upon Helicobacter infection is still not clear. In this paper we characterized TFFs expression, with particular attention to TFF1, under Helicobacter infection in gastric cell lines. A mouse model was used to distinguish TFF1 mRNA expression between acute and chronic stages of Helicobacter infection. Our results show that TFF1 expression is induced in infected cells; in addition, the inflammatory response upon Helicobacter infection is inversely associated to pre-existing TFF1 protein levels. In infectedmice, TFF1 is initially upregulated in gastric antrum in the acute phase of infection, along with IL-1b and IL-6. Then, expression of TFF1 is gradually silenced when the infection becomes chronic and IFN-g, CXCL5, and CXCL15 reach higher levels. Our data suggest that TFF1 might help cells to counteract bacteria colonization and the development of a chronic inflammation